title: Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease.
abstract: CF (cystic fibrosis) is caused by mutations in CFTR (CF transmembrane conductance regulator), which cause its mistrafficking and/or dysfunction as a regulated chloride channel on the apical surface of epithelia. CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remain unclear. In the present study, we evaluated the mechanism of action of one small-molecule modulator, VRT-532, identified as a specific modulator of CF-causing mutants. First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the Wt (wild-type) CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis.
authors.affiliation: Programme in Molecular Structure and Function, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
authors.forename: Stan
authors.initials: S
authors.lastname: Pasyk
authors.forename: Canhui
authors.initials: C
authors.lastname: Li
authors.forename: Mohabir
authors.initials: M
authors.lastname: Ramjeesingh
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003408:Cresols / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology*; D018411:Spodoptera
chemical_list: D003408:Cresols; D007454:Iodides; D011720:Pyrazoles; C535089:VRT 532; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D005998:Glycine